Differentiation in the Gram-negative, aquatic bacterium C. crescentus, occurs as the direct result of asymmetric cell division to produce a nonmotile stalked cell and a new, motile swarmer cell. The differentiated surface structures that characterize the swarmer cell are assembled in the cell envelope at the incipient swarmer pole of the dividing stalked cell in a discrete temporal sequence during the cell cycle. These developmental events, which include flagellum biosynthesis, bacteriophage receptor formation, and pili formation, are triggered by internal signals originating from the cell division cycle. Successive stages of the cell division cycle are required as checkpoints for these developmental events. The long term goal of our studies is to decipher the genetic program that controls polar morphogenesis in Caulobacter. To achieve this, our work has focused primarily on the flagellum, whose assembly requires approximately 50 flagellar (fla) genes. These genes have been assigned to four classes (I-IV) within a regulatory hierarchy in which class I genes are at the top of the hierarchy and regulate transcription of class II-IV genes below. The focus of this proposal is to understand how the cell cycle regulates initiation of this transcriptional cascade and the molecular mechanisms that control transcriptional specificity within the cascade. Our specific aims include: l. Isolate sigma factors and regulatory proteins required for transcription from the class II promoters located near the top of the Caulobacter crescentus fla gene hierarchy. 2. Identify and isolate class I genes responsible for coupling initiation of fla gene transcriptional cascade to DNA replication in the cell cycle. 3. Examine the dependence of FlbD activity on other class II fla genes for the in vivo activation of class III and repression of class IIB genes. 4. Identify the mechanism that mediates the transition from expression of class III to class IV genes at the bottom of the fla gene hierarchy. We propose experiments based on genetic, molecular, and biochemical approaches to achieve these goals.